Fermentative method for the production of l-glutamic acid



United States Patent s 335 065 FERMENTATIVE Mrirubn FOR THE PRODUC- TIONor L-GLUTAMIC ACID Katsunobu Tanaka, Machida-shi, Kazuo Oshima, Tokyo,

3,335,065 Patented Aug. 8, 1967 ice Gram positive (in an old culture,Gram negative of about 50% is recognized).

(B) CULTURAL CHARACTERISTICS and Kenichiro Takayama, Mitaka-shi, Japan,assignors Agar p g y Weak g circular, Smooth, to Kyowa Hakk Kogyo (30.,Ltd Chi oda-ku, Tokyo, slightly raised to flat, entire pale yellow,opaque, dull, Japan, a corporation of Japan slightly dry.

N0 P q -F l 1965, 433,517 (2) Agar slant: moderate to scanty growth,filiform, Clalms Priority Pg f Japan, 1964, raised to flat, dull,smooth, opaque, pale yellow,

butyrous. 7 Chums (CL 195.30) (3) Broth: slightly turbidic, flocculentsediment, no odor. The present invention relates to the fermentativeprogar ta l: best at Stop, filiform. duction, i.e. with the aid ofmicroorganisms, of L-glu- Glucose nutrient agar: scanty I10 B (C)PHYSIOLOGICAL CHARACTERISTICS It 18 known that many microorganismswhich, when cultured, produce L glutamic acid from carbohydrates, (1)Optlmum temperature! 'g growth may also produce L-glutamic acid fromacetic acid as the 37 sole carbon source. However, such fermentativeproduc- (2) P P P tion of L g1u/[amic acid by known glutamic acid produc(3) Relat on to free oxygen: aerobic to faculative ing bacteria fromacetic acid, as the raw material, is anaqrobwbound up with thedisadvantage of low fermentation yield (4) L1tmu mllk? P changedincomparison with the yields obtained when using carbo- (5) Gelatm: not qhydrate as raw material. The fermentative conversion of (6) HydrogenSulfide: not formedacetic acid to L-glutamic acid with the aid of thesaid (7) Indole: not formedbacteria is thus of no practical significanceand cannot (8) f not hydrolysedbe used on a commercial scale. (9)Nltmte: not d c The primary object of the present invention is the em-(10) catalase: 'Pf bodirnent of a commercial process for thefermentative (11) Urease: posltlveproduction of L-glutamic acid fromacetic acid, as such (12) Acetylmethylcafblnoli P formedor in the formof its alkali metal or ammonium salt, as (13) lf Red test: negatlvetheraw material carbon source. Briefly stated, this object (14) Acld 13 notformed from p yf is achieved by the use of a special novel strain,namely Large 0f L-glll'lamlc acid are Produced Corynebacteriumacetoglutamicum (KY N0. 3513), which from acetlc has been isolated f then near Tokyo, Japan (16) When glucose or acetic acid is used as a.carbon and which is also obtained from animal faces. The micro- 4 2 Q4is not utilized as Sole nitrogen Source organism is a Corynebacterium,as will hereinafter be Huckers W demonstrated. Its epithet (speciesname) is based upon its (17) cellulose 15 not decomposed property ofproducing Lgmtamic acid in significant The present bacterium wasclassified according to quantities Bergeys Manual of DetermmativeBacteriology, 7th hi property f producing large amounts f Lghuamic 0edition. ThlS stram belongs to family Corynebacteriaceae acid fromacetic acid in the fermentation liquid, i.e. in because it is GramPositive, not Spoufated, aerobic and the acetic acid-containing nutrientmedium employed for Tod Shape with granules- Furthermore, it is p p toaerobically culturing the said Corynebacterium acetoclassify it in gecorynebacterium because it is Gram glutamicum (KY No. 3513) solves theproblem of h positive, non-motile, does not decompose cellulose andfermentative production, on a commercial scale, of L- isolated fromanimal faces. glutamic acid from the relatively inexpensive raw ma-Points of difference in related strains are as follows:

Corynebacterium Corz/nebacterium Present bacterium pseudodiphequitheriticum Color in nutrient agar- Pale yellow Gray or crearn Tan toyellow or pink to red. Growthinnutrient agar Slightly weak; MoistUsually moist.

slightly dry. Optimum temperature 25-30 C 37 0- 25-37 0. Growth 011 theaddition of glucose Inhibited Stimulated.

(A) MORPHOLOGICAL CHARACTERISTICS Shape of bacterium: usually short rodswith round ends and sometimes ellipsoidal or club-shape. Occurring insingle or pairs.

Size: 0.8 to 1.2 x 1.2 to 3.0a.

Motility: non-motile.

Metachromatic granules observed: branching cells rarely recognized.

A culture of Corynebacterium acetoglutamicum (KY No. 3513) is on depositunder accession No. ATCC 15806, at American Type Culture Collection,Rockville, Md.

In the process according to the present invention, the raw materials:acetic acid, sodium acetate, potassium acetate, ammonium acetate, etc.are used as assimilable carbon source, while the assimilable inorganicor organic nitrogen sources conventionally employed in the fermentativeproduction of gluta-micby meansof usual glutarnic acid-producingbacteria are here also used as nitrogen source.

In carrying out the culturing according to the present invention, thenutrient medium (culture medium) containing the acetic acid oraforementioned salt thereof as sole assimilable carbon source andalsocontaining a nitrogen source as precedingly defined as well as the usualinorganic salt, has its pH adjusted to 6.0 to 7.0, and is thensterilized. The strain employed according to the present invention,Cov'ynebacterium acetoglutamicum (KY No. 3513), is then inoculated intothe said culture medium and incubated at 25 to 30 C. under aerobicconditions. During the culturing period, the pH of the culture medium isadvantageously adjusted to 6.8 to 7.8 by the addition thereto of aqueoussolution of acetic acid and ammonium acetate. The fermentation iscompleted when the production of L-glutamic acid reaches its maximumusually in 2 or 3 days. The fermentation liquor is then passed throughion exchange resin, in per se known manner, whereupon the L-glutamicacid is adsorbed. The L-glutamic acid is then extracted with aqueousalkali, concentrated, and cooled. Crystals of L-glutamic acid separate,are isolated and recrystallized. The recovery or harvesting of theL-glutamic acid from the fermentation liquor is per se no part of thepresent invention, and may be carried out in any manner known in priorfermentative processes for the production of L-glutamic acid. (Any otherknown procedure for recovering L-glutamic acid from a fermentationmedium in which it was produced, can also be employed.)

The following illustrative but non-limitative examples of presentlypreferred embodiments of the invention further explain the presentinvention. The invention extends no only to the use of the aforesaidmicroorganism [Corynebacterium acetoglutamicum (KY No. 3513)] but alsoto mutant strains thereof with the characteristic of the said bacterium,and produced e.g. by mutation under the action of ultra-violet rays,X-rays, etc.

Example 1 A culture medium consisting of Grams Sodium acetate 500Ammonium sulfate 200 Meat extract 20 Calcium carbonate 300 KH P-OMgSO4'7H2O 2 Urea 30 and Water, ad. 10 liters.

is adjusted to pH 7.0 with sodium hydroxide solution. Corynebacteriumacetoglutamicwm (KY No. 3513) (ATCC No. 15806) is inoculated in thethus-obtained fermentation medium, and aerobic shaking culture iscarried out at 30 C. grams of L-glutamic acid per liter is accumulatedin 72-hour culture. 10 liters of the fermentation liquor thus obtainedis passed, in per se known manner, through cation exchange resin wherebythe L-glutamic acid is adsorbed thereon. The adsorbed L-glutamic acid isthen eluted from the resin with dilute aqueous ammonia. Crystallizationof the L-glutamic acid from the efiluent containing the same insolution, in conventional manner, yields 120 grams of L-glutamic acid.

Example 2 Corynebacterium acetoglutamicum (KY No. 3513) (ATCC No. 15806)is inoculated into a culture medium containing, per 3 liters,

Grams Sodium acetate 6O Ammonium sulfate 30 KH PO 1.5 K HPO 1.5 MgSO -7HO 1.5 FeSO -7H O 0.6 MnSO '4H O 0.060 Meat extract 6 Peptone 6 theremainder being water, and the medium being adjusted to pH 6.0 to 7.0.Aerobic shaking culture is carried out at 27 to 30 C. 8 grams ofL-glutamic acid per liter is accumulated in 40-hour culture.

Purification after the manner described in Example 1 yields 19.8 gramsof purified crystals of L-glutamic acid.

Example 3 Corynebacterium acetoglutam icum (KY No. 3513) (ATCC No.15806) is inoculated into a culture medium consisting of Grams Sodiumacetate 200 Peptone Meat extract 50 Yeast extract 50 Sodium chloride 25and Water, ad. 10 liters.

at pH 6.5 to 7.5. Incubation is carried out at 30 C. for 24 hours withshaking under aerobic conditions.

Thereafter, 10 milliliters of the thus-obtained 54-hour culture isinoculated into an aqueous fermentation medium containing, per 10 litersof medium,

Sodium acetate kil0 grams- 2 Ammonium sulfate do 1 KH PO grams 50 K HPOdo 50 MgSO '7H O dO FeSO -7H O do 20 M11804 dO 2 the remainder beingwater. Incubation is then carried out at 30 C. with shaking underaerobic conditions. During this cultivation, there is gradually added anaqueous solution containing 4 parts of acetic acid and 1 part ofammonium acetate, the pH of the fermentation liquor being thusmaintained at pH 6.8 to 7.8. 50 grams of L-glutamic acid per liter arethus accumulated in 48-hour culture. The fermentation yield is thusequivalent to 45% by weight relative to the weight of acetic acidemployed.

Purification after the manner set forth in Example 1 yields 4.0kilograms of L-glutamic acid.

Where purification of the L-glutamic acid produced according to thepresent fermentation process is carried out with cation exchange resin,use may be made e.g. of a commercially available resin cation exchangematerial of the acid regenerated type. Such exchange resins are obtainedby condensing aldehydes such as formaldehyde with phenols or withphenol-sulfonic acids, etc. A sulfonated polystyrene type cationexchange resin may also be used; these are commercially available underthe name of Amberlite.

What is claimed is:

1. In a process for producing L-glutamic acid by cultivating anL-glutamic acid-producing microorganism in a nutrient medium containingacetic acid as sole assimilable carbon source, and no NH H PO untilL-glutamic acid has accumulated and recovering the so-accumulatedL-glutamic acid, the improvement according to which the microorganism isCorynebaclerium acetoglutamicum (ATCC No. 15806), whereby enhancedamounts of L- glutamic acid are produced.

2. In a process for producing L-glutamic acid by cultivating anL-glutamic acid-producing microorganism in a nutrient medium containingacetic acid as sole assimilable carbon source, and no NH H PO untilL-glutamic acid has accumulated and recovering the so-accumulatedL-glutamic acid, the improvement according to which the microorganism isa mutant of Corynebacterium acetoglutamicum (ATCC No. 15806), wherebyenhanced amounts of L-glutamic acid are produced.

3. In a process for producing L-glutamic acid by cultiva ing ll L-g'utamic acid-producing microoganism in a nutrient medium containing amember selected from the group consisting of alkali metal acetate andammonium acetate as sole assimiliable carbon source, and no NH H POuntil L-glutamic acid has accumulated and recovering the so-accumulatedL-glutamic acid, the improvement according to which the microorganismsis Corynebacterium acetoglutwmicum. (ATCC No. 15806), whereby enhancedamounts of L-glutamic acid are produced.

4. A method of producing L-glutamic acid which comprises cultivatingunder aerobic conditions Corynebacterium acetoglutazmicum (ATCC No.15806) in an aqueous nutrient medium containing a source of assimiliablenitrogen, no NH H PO and, as sole source of assimilable carbon, aceticacid until maximal production of L- gl utamic acid has been achieved,and recovering such L-glutamic acid from the fermentation broth.

5. A method of producing L-glutamic acid which comprises cultivatingunder aerobic conditions Corynebacteriwm acetogluta m'icum (ATCC No.15806) in an aqueous nutrient medium containing a source of assimilablenitrogen, no NH H PO and, as sole source of assimilable carbon, a memberselected from the group consisting of alkali metal acetate and ammoniumacetate runtil maximal production of L-glutamic acid has been achieved,and recovering such L-glutamic acid from the fermentation broth.

6. In a process for producing L-glutamic acid by cultivating anL-glutamic acid producing microorganism in a nutrient medium containingacetic acid as sole assimilable carbon source, until L-glutamic acid hasaccumulated, and recovering the so-accumulated L-glutamic acid, theimprovement according to which the microorganism is Corynebacteriumacetoglutamicum (ATCC No. 15806), whereby enhanced amounts of L-glutamicacid are produced.

7. A method of producing L-glutamic acid which comprises cultivatingunder aerobic conditions Corynebacteriwm acetoglwtwmicmn (ATCC No.15806) in an aqueous nutrient medium containing as sole source ofassimilable carbon, a member selected from the group consisting ofacetic acid, alkali metal acetate, and ammonium acetate, untilL-glutamic acid has accumulated, and recovering such L-glutamic acidfrom the fermentation broth.

References Cited UNITED STATES PATENTS 3,117,915 1/1964 Shiio et a1.195-30 A. LOUIS M'ONACELL, Primary Examiner.

L. M. SHAPIRO, Assistant Examiner.

6. IN A PROCESS FOR PRODUCING L-GLUTAMIC ACID BY CULTIVATING ANL-GLUTAMIC ACID PRODUCING MICROORGANISM IN A NUTRIENT MEDIUM CONTAININGACETIC ACID AS SOLE ASSIMILABLE CARBON SOURCE, UNTIL L-GLUTAMIC ACID HASACCUMULATED, AND RECOVERING THE SO-ACCUMULATED L-GLUTAMIC ACID, THEIMPROVEMENT ACCORDING TO WHICH THE MICROORGANISM IS CORYNEBACTERIUMACETOGLUTAMICUM (ATCC NO. 15806), WHEREBY ENHANCED AMOUNTS OF L-GLUTAMICACID ARE PRODUCED.